1. Field of the Invention
The invention relates to the family of cytosolic immunosuppressant drug binding proteins termed immunophilins. More specifically, this invention relates to a novel immunophilin.
2. Background Information
Cyclosporine, FK-506 and rapamycin are drugs of clinical importance in suppressing organ transplant rejection by suppression of the immune system. Although the mechanism of action of these drugs is not yet understood, it is clear that an initial step in this mechanism consists of the high affinity binding of the drug to water-soluble cytoplasmic (cytosolic) binding proteins termed immunophilins.
Several immunophilins with various drug binding specificities have been identified. Certain of them have been purified to homogeneity, and completely or partially sequenced. At least five specific binding proteins for the macrolides FK-506 and rapamycin are known. The most abundant of these is the immunophilin termed FKBP-12, a protein of molecular mass of about 11-12 kDa that has been purified to homogeneity (Siekierka et al., U.S. Pat. No. 5,109,112; Schreiber et al., WO 91/04321; Lane et al., J. Prot. Chem., 10:151 (1991); Soldin, U.S. patent application Ser. No. 07/782,761).
A membrane-associated human FK-506 and rapamycin binding protein of molecular mass of about 13 kDa and termed FKBP-13 has been cloned from a human carcinoma cDNA library and sequenced; it has a 51% nucleotide sequence identity and 43% amino acid sequence identity to FKBP-12 and, like FKBP-12, exhibits rotamase activity (Jin et al., Proc. Natl. Acad. Sci. (USA), 88:6677 (1991)). A yeast membrane-associated FKBP-13 has also been identified, purified and partially sequenced; it binds both FK-506 and rapamycin, albeit with a substantially lower affinity than exhibited by FKBP-12, and in common with FKBP-12, possesses peptidylprolyl cis-trans isomerase (rotamase) activity (Nielsen et al., Proc. Natl. Acad. Sci. (USA), 89:7471 (1992)).
A 25 kDa high affinity rapamycin-binding protein has been identified and cloned from calf thymus, brain and spleen, and overexpressed in E. coli. This protein, which, like FKBP-12 and FKBP-13, exhibits peptidylprolyl cis-trans isomerase (rotamase) activity, shares identify with FKBP-12 (44%) and FKBP-13 (47%) in the C-terminal 97 amino acid region (Jin et al., J. Biol. Chem., 267:10942 (1982); Galat et al., Biochemistry, 31:2427 (1992)).
Soldin et al. have purified to homogeneity and partially sequenced a low-abundance cytosolic immunophilin of a molecular mass of about 52 kDa. This protein has an unusually broad specificity in that it binds the cyclic undecapeptide cyclosporine, as well as the macrolides FK-506 and rapamycin, albeit at apparently different binding sites (Donnelly et al., Transplant. Proc., 23:2886 (1991); Soldin, U.S. patent application Ser. No. 07/782,761). This protein is devoid of rotamase activity, but inhibits cyclic AMP-activated protein kinase activity.
Characterizing immunophilins is important, not only for the elucidation of the mechanism of immunosuppressive action of FK-506 and rapamycin, but also because such proteins are valuable in developing quantitative binding assays for the circulating concentration of these drugs and their biologically active metabolites and derivatives, and in developing affinity systems for the isolation of immunosuppressive drugs from natural sources.
We have discovered, purified to homogeneity, and partially sequenced a novel cytosolic immunophilin that specifically binds FK-506 and rapamycin, but not cyclosporine A. This discovery is described below.